"Noise" - random fluctuation of cellular components - is a fundamental aspect of life at the single cell level. Noise plays a dual role in biological systems: On the one hand, it allows cells to make random decisions biased by external conditions. For instance, by sporulating in response to stress individual cells make probabilistic 'bets' about their future environment. On the other hand, noise can interfere with developmental processes that depend on precise genetic regulation. Intracellular noise has recently been detected and quantified in simple synthetic circuits. Here we propose to analyze noise directly within natural genetic circuits that make probabilistic cell-fate decisions and undergo precise developmental processes. Bacillus subtilis presents a unique opportunity to do so: It uses well-characterized genetic circuits to probabilistically initiate the differentiation programs of competence and sporulation. It also undergoes a tightly-coordinated, noise-suppressing developmental process during sporulation. The goal of this research is to understand how B. subtilis gene circuits amplify noise to probabilistically regulate competence events and sporulation initiation, and how they suppress noise to generate an ordered sequence of events during sporulation. We will apply quantitative time-lapse fluorescent microscopy techniques to observe gene circuit dynamics in single cells, and 're-wire' circuits to test specific predictions. The overall approach will be driven by mathematical models of underlying gene circuits. We will specifically address three problems: (1) In the case of competence regulation, we will test the hypothesis that noise- driven excitability generates probabilistic and transient competence episodes. (2) In the case of sporulation initiation, we will test the hypothesis that nested positive feedback loops, together with noise, generate temporal variability in the decision to initiate sporulation. (3) In the developmental process of sporulation, we will determine how noise is suppressed in wild-type cells but determines the fate of partially penetrant (PP) mutants (mutants in which some cells successfully complete sporulation, while others die). The circuit-level strategies identified in this model system are likely to operate in more complex organisms that undergo differentiation and development. Relevance to Public Health: The spread of infectious diseases depends on probabilistic activation of alternative genetic programs, such as competence, sporulation, and antibiotic persistence in bacteria, and latency in viruses. This proposal will address the mechanism by which individual cells randomly enter these alternate states. In addition, the proposal will investigate the mechanisms leading to the partial penetrance (occurring only in some affected individuals) of mutations. Partial penetrance is also found in human diseases. [unreadable] [unreadable] [unreadable]